The citrate carrier, Slc25a1, is a nuclear encoded protein that localizes in the inner mitochondrial membrane, where it serves an important function in promoting the flux of the lipid precursor citrate in the cytoplasm. Slc25a1 gene alterations play a key role in the pathogenesis of a variety of developmental disorders. Heterozygous deletions of the gene are hallmark DiGeorge and Velo-Cardio-Facial Syndrome, while missense Slc25a1 gene mutations give raise to combined D-2-/L-2-hydroxyglutaric aciduria and to a variety of still incompletely classified disorders. The clinical spectrum of manifestations associated with Slc25a1 dysfunction consists of severe craniofacial abnormalities, brain abnormalities, respiratory insufficiency, epileptic encephalopathy, as well as metabolic dysfunction characterized by lactic acidosis and by urinary excretion of two abnormal products of the tricyclic acid (TCA) cycle, D2-L2 hydroxyglutaric acids. The severity of manifestations seen in patients harboring Slc25a1 mutations is variable, spanning from very moderate to extremely severe, leading to early death within the first month of age. Unfortunately, there is no therapy for syndromes associated with Slc25a1 deficiency, due to the lack of full understanding of the exact molecular mechanisms by which Slc25a1 operates. To address this gap in knowledge we have developed the first murine and zebrafish models of Slc25a1 deficiency and we find that in both systems Slc25a1 loss results in pre- and perinatal lethality and in pathologic features that recapitulate salient aspects of human Slc25a1 deficiency. We further identify and novel activity of Slc25a1 consisting in regulation of mtDNA homeostasis. Molecular and metabolic analyses demonstrate that tissues and cells derived from Slc25a1-/- mice exhibit abnormal TCA cycle flux, respiratory deficit and upregulation of several metabolic pathways, including lipid synthetic pathways. This suggests that the pathogenesis of Slc25a1 disorders may be due to harmful upregulation of compensatory metabolic routes, at least in part consequent to mitochondrial dysfunction. We propose to test this idea with two specific aims. In Aim 1 we will map the metabolic and molecular changes due to Slc25a1 deficiency and we will define the mechanisms by which Slc25a1 affects mitochondrial activity. In Aim 2 we will compare the effects of Slc25a1 loss versus human-associated Slc25a1 mutations in the model organism zebrafish. These studies will be pivotal for the identification of the molecular alterations associated with Slc25a1 dysfunction which, in turn, could strategically guide future therapeutic efforts.